体外共培养 DiI 标记大鼠骨髓间充质干细胞和聚己内酯膜制作细胞片的实验研究。

Experimental study on co-culture of DiI labeled rat bone marrow mesenchymal stem cells and polycaprolactone film in vitro to make a cell patch.

机构信息

Department of Cardiac Surgery, Beijing Anzhen Hospital, Capital Medical University, Beijing, China.

Department of Ultrasound, The Third Medical Center of PLA General Hospital, Beijing, China.

出版信息

Biomed Mater Eng. 2022;33(4):269-277. doi: 10.3233/BME-211312.

DOI:10.3233/BME-211312

PMID:34719477

Abstract

BACKGROUND

In stem cell therapy, due to the lack of an effective carrier, a large number of transplanted stem cells are lost and die. Therefore, finding a suitable carrier has become a further direction of stem cell therapy.

OBJECTIVE

In research on the co-culture of polycaprolactone (PCL) with 1,1'-Dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate (DiI) labeled bone marrow mesenchymal stem cells (BMSCs), we observe the effect of materials on the growth and proliferation of DiI labeled stem cells, and the effect of DiI labeling on patch preparation, so as to find a kind of biomaterial suitable for the growth and proliferation of BMSCs, and find a suitable cell carrier for stem cell therapy of myocardial infarction and in vivo tracing.

METHODS

Clean grade Sprague Dawley rats were selected as experimental objects, BMSCs were isolated and cultured, and the surface markers were identified by flow cytometry. After the BMSCs were cultured for 3 passages, the BMSCs were stained with DiI dye, and the BMSCs DiI and PCL biomaterial film were co-cultured. After 24 hours, the cell growth was observed under fluorescence microscope, and fixed for scanning under electron microscope. The cell proliferation was detected by CCK-8 at 1, 4, 7, 10 days of culture. The measurement data conforming to normal distribution are expressed in the form of mean ± standard deviation (X¯± s). One way ANOVA was used for comparison among groups, LSD analysis was used for pairwise comparison. The difference was statistically significant (P < 0.05).

RESULTS

BMSCs were strongly positive for CD90, CD44H, but negative for CD11b/c, CD45. Under fluorescence microscope, BMSCs DiI showed red light, fusiform or polygonal. Under the scanning electron microscope, the cell patch formed by co-culture of PCL film and DiI-BMSCs had a large number of cells on the surface and normal cell state. CCK-8 assay showed that the OD value on the first day was 0.330 ± 0.025; The OD value was 0.620 ± 0.012 on the 4th day, 1.033 ± 0.144 on the 7th day and 1.223 ± 0.133 on the 10th day. There was significant difference among the time points (P < 0.05).

CONCLUSIONS

The cell patch made of PCL film and DiI labeled BMSCs can survive and proliferate on the surface, so it can be used as a scaffold material for stem cell therapy in vivo.

摘要

背景

在干细胞治疗中，由于缺乏有效的载体，大量移植的干细胞会丢失和死亡。因此，寻找合适的载体已成为干细胞治疗的进一步方向。

目的

在聚己内酯（PCL）与 1,1'-二辛基-3,3,3',3'-四甲基吲哚羰花青高氯酸盐（DiI）标记骨髓间充质干细胞（BMSCs）共培养的研究中，观察材料对 DiI 标记干细胞生长和增殖的影响，以及 DiI 标记对贴剂制备的影响，从而找到一种适合 BMSCs 生长和增殖的生物材料，并找到一种适合心肌梗死干细胞治疗和体内示踪的细胞载体。

方法

选用清洁级 Sprague Dawley 大鼠作为实验对象，分离培养 BMSCs，流式细胞术鉴定表面标志物。BMSCs 培养 3 代后，用 DiI 染料染色，将 BMSCs-DiI 与 PCL 生物膜共培养。24 小时后，荧光显微镜下观察细胞生长情况，电镜下固定扫描。培养 1、4、7、10 天时，用 CCK-8 检测细胞增殖。符合正态分布的计量资料以均数±标准差（X¯±s）表示。多组间比较采用单因素方差分析，组间两两比较采用 LSD 分析。差异有统计学意义（P<0.05）。

结果

BMSCs 对 CD90、CD44H 呈强阳性，对 CD11b/c、CD45 呈阴性。荧光显微镜下，BMSCs-DiI 呈红色，梭形或多边形。扫描电镜下，PCL 膜与 DiI-BMSCs 共培养形成的细胞贴片中，表面有大量细胞，细胞状态正常。CCK-8 检测显示，第 1 天 OD 值为 0.330±0.025；第 4 天 OD 值为 0.620±0.012，第 7 天 OD 值为 1.033±0.144，第 10 天 OD 值为 1.223±0.133。各时间点间差异有统计学意义（P<0.05）。